The present invention is directed to sample handling. More particularly, certain embodiments of the present invention provide sample containers adapted for acoustic ejections and analyses and methods thereof. Merely by way of example, the invention has been applied to biological samples. But it would be recognized that the invention has a much broader range of applicability, such as preservation of liquid sample within a sample container.
It is often desired to take a biological sample (e.g., a human blood sample) contained in an individual sample holder and to transfer it to one or more well plates or other objects appropriate for carrying out reactions with the biological sample (e.g., onto test strips). Usually, a single biological sample (e.g., a single human blood sample) may be divided up among a number of these downstream containers in order to be subjected to a wide variety of different tests. Many biological samples also include a plurality of components that may be separable and preferentially subjected to tests as separable components. For example, blood samples in tubes are commonly centrifuged and stratified into multiple layers, with plasma located at the top, red blood cells at the bottom and other blood components in between. Furthermore, many biological samples may degrade during storage, so it is desirable to adapt the sample holder to create an environment to preserve the biological samples.
Some important considerations for the handling of biological samples include: ability to obtain a number of measurements from a single extracted sample (e.g., a single blood draw); generating no waste in the sample transfer; providing a proportionate amount of fluid, particulates and cells with a transfer and overcoming challenges in achieving such transfer at small volumes; enabling various types of diagnostics that can benefit from consistent deliveries of small-volume samples; elimination of manual pipetting and associated wastes and interactions with tips, sharps, capillaries, and needles, in order to improve lab safety; reducing the training needed for lab technicians to achieve high-quality small-volume sample transfers; and/or using the same container for blood collection, storage and as a source for transfer.
Acoustic ejection has been known for a number of years as a way of performing transfers of biological samples. For example, in a typical setup for acoustic ejection, a piezoelectric transducer is driven by a waveform chosen by a controller and in response generates acoustic energy. The acoustic energy often is focused by an acoustic lens, and coupled to a reservoir containing fluid through an acoustic coupling medium (e.g., water). If the focused energy has a focal point inside a fluid in the reservoir and close to a free surface of that fluid, a droplet may be ejected. Droplet size and velocity can be controlled by the chosen waveform as mentioned above.
In some embodiments, the transducer is movable in one or more directions (e.g., in the “z direction”) that is roughly perpendicular to the free surface of the fluid. The movement can take place under the control of the controller. Some acoustic instruments for high-throughput use rely on an active control of the transducer position relative to the reservoir and address the multiplicity of wells in microplates. Often, the adjustment of the transducer position involves sending a motion command to a motion controller which then initiates movement in one or more directions (e.g., along one or more axes). For example, motion in the horizontal plane (e.g., in the “x direction” and/or in the “y direction”) aligns the transducer with the selected reservoir, and motion in the vertical direction (e.g., in the “z direction”) is used both to audit the reservoir and to focus for droplet transfer. In another example, positioning of the transducer to achieve the proper focus for droplet ejections can be responsive to data collected from an acoustic audit. Additionally, U.S. Pat. Nos. 6,938,995 and 7,900,505 are incorporated by reference herein for all purposes. When the motion is complete, the controller can notify the system that the transducer and the reservoir are now in the proper position for the next step in the process. In some contexts, it is desirable to accomplish acoustic ejections of selected regions within a single sample that have been separated, and such ejections may be facilitated by motion of the transducer in the horizontal plane. Moreover, U.S. Pat. No. 6,666,541 is incorporated by reference herein for all purposes.
Beyond transfer of simple fluid, focused acoustic energy recently has been used in applications involving biological matters such as living cells. For example, focused acoustic radiation has been used to manipulate and sort cells. U.S. Patent Application Publication Nos. 2002/0064808 and 2002/0064809 and U.S. Pat. Nos. 6,893,836 and 6,849,423 are incorporated by reference herein for all purposes. In another example, containers for sample collection are also widely used. The conventional containers (e.g., containers used in extraction and/or storage of samples) usually are not adapted for use in acoustic transfer.
Additionally, methods for separation of samples within sample containers are well known. For example, blood samples are commonly centrifuged to separate components into layers. Often, such separation is performed in order to access a particular component for analysis. In another example, a stratum from a normal blood sample, from top to bottom, includes plasma, platelets, non-granulocytes (e.g., lymphocytes and monocytes), granulocytes (e.g., basophils, eosinophils, and neutrophils) and then red blood cells (e.g., reticulocytes and erythrocytes).
Moreover, methods for improving homogeneity of samples within sample containers are also well known. For example, blood samples are commonly inverted manually (e.g., for twenty times) to agitate the samples and re-suspend the cells. In another example, automated systems for sample handling can perform a similar function by rocking and/or rotating sample containers.
Additionally, methods to prevent degradation of samples are well known. Blood samples often are collected into containers that already have reagents inside to preserve the samples. For example, ethylenediaminetetraacetic acid (EDTA), a strong chelator of metal ions, is added to eliminate or reduce the activity of metalloproteases in solution because such activity can degrade peptide biomarkers. Additionally, EDTA can also prevent or reduce coagulation of the blood samples by binding to calcium ions and preventing the coagulation cascade from occurring. In another example, sodium fluoride is added to sample containers (e.g., blood collection tubes) in order to stop degradation of glucose in the blood samples. In yet another example, these additives (e.g., EDTA and sodium fluoride) are present in the sample containers as a solution, a dried residue and/or a non-covalent coating (e.g., spray-coating) on interior surfaces of the containers.
Often, these additives can become integral to the blood samples after the samples enter the containers, and these additives may have a negative impact on downstream assays. Therefore, blood samples often are collected in multiple sample containers (e.g., tubes) that contain specific additives that are compatible with respective downstream analyses.
Furthermore, the sample containers (e.g., blood collection tubes) with additives often require that the blood samples and the additives be mixed through partial or complete inversion of the containers once the containers are filled with blood samples. The efficacy of mixing by inversion or semi-inversion usually is dramatically decreased as the volume of the blood sample and the size of the sample container are reduced. Efficacy loss can be due to inability of the container motion to be strong enough to overcome the adhesion of the sample to the container surfaces, or inability of the change of container in orientation with respect to gravity to be strong enough to overcome the adhesion of the sample to the container surfaces. The reduction in efficacy may lead to a delay in the role of the additives as preservatives of certain biological markers.
Researchers also have shown that endogenous enzymes (e.g., proteases and peptidases) in blood samples can degrade and destroy peptide and protein constituents, which are important biomarkers of the blood samples. Hence some blood collection tubes have been introduced with additives that can inhibit various proteases.
Therefore, there is a need for acoustic ejection systems and sample containers that can in combination significantly simplify handling of biological samples and are amenable to miniaturization.